Herpes simplex virus thymidine kinase (HSVtk) gene transfer followed by ganciclovir administration is a common strategy for experimental cancer therapy. To evaluate the feasibility of using the human calcitonin promoter to target medullary thyroid carcinoma (MTC), we developed adenovirus vectors containing Escherichia coli beta-galactosidase gene under the control of the CALC-I promoter (AdCTlacZ), or the human cytomegalovirus promoter (AdCMVlacZ). Beta-galactosidase activity driven by the CALC-I promoter was higher than by the CMV promoter in rat MTC cells after infection with adenovirus vectors. AdCTlacZ induced an equal or lower expression level of beta-galactosidase in TT (human MTC), T98G, Cos1, HepG2, and HeLa cells compared with AdCMVlacZ. To inhibit the growth of MTC cells, we developed two adenovirus vectors, AdCMVtk carrying HSVtk driven by the cytomegalovirus promoter and AdDCTtk containing a human CALC-I minigene under the control of the CALC-I promoter. HSVtk is fused to a portion of calcitonin coded in exon 4 to direct cell-specific regulation of splicing. All cell lines infected with AdCMVtk were rendered sensitive to ganciclovir, whereas T98G and Cos1 cells infected with AdDCTtk were not affected. Cell killing was also observed in HeLa, HepG2, rat MTC and TT cells infected with AdDCTtk.